Gas fermentation for the production of protein or feed

ABSTRACT

The invention provides animal feed comprising microbial biomass and methods of producing animal feed by culturing a microorganism to produce microbial biomass. In particular, the invention relates to animal feed produced by fermentation of a gaseous substrate comprising one or more of CO, CO 2 , and H 2 , especially by a Gram-positive, anaerobic, and/or  Clostridium  microorganism.

BACKGROUND OF THE INVENTION

Single cell protein (SCP) refers to microbial biomass for use inprotein-rich human and animal feeds, often replacing conventionalsources of protein supplementation such as soymeal or fishmeal.

Large-scale production of microbial biomass has many advantages overtraditional methods for producing proteins for food or feed. Forexample, microorganisms have high growth rates, can be geneticallymodified to tailor amino acid composition, have high protein content,and can utilize a broad spectrum of carbon and energy sources. Moreover,bioconversion of agricultural and industrial wastes to protein-rich feedstocks has an additional benefit of making the final product cheaper,which also offsets the negative cost value of wastes used as substrateto yield SCP. Further, use of SCP renders feed production less dependentupon land resources and relieves pressure on agriculture.

Algae, fungi, and bacteria are the chief sources of SCP (Ravindra,Biotechnol Adv, 18: 459-479, 2000). Bacterial species previously usedfor SCP include Methylophilus methylotrophicus (Imperial ChemicalIndustries), Methylophilus clara (Hoechst), and Methylophilus methanica(Norsk Hydro). However, each of these microorganisms are Gram-negativeand aerobic and consume methanol as a carbon source. Accordingly, thereremains a need for additional animal feeds comprising microbial biomassfrom different strains and grown on different carbon sources.

SUMMARY OF THE INVENTION

The invention provides animal feed comprising microbial biomass and atleast one excipient. Generally, the microbial biomass comprises amicroorganism grown on a gaseous substrate, such as a gaseous substratecomprising one or more of CO, CO₂, and H₂.

The microorganism may be Gram-positive, acetogenic, carboxydotrophic,and/or anaerobic. Generally, the microorganism is a member of the genusClostridium, such as a microorganism that is or is derived fromClostridium autoethanogenum, Clostridium ljungdahlii, Clostridiumragsdalei, or Clostridium coskatii. In a preferred embodiment, themicroorganism is Clostridium autoethanogenum deposited under DSMZaccession number DSM23693. In certain embodiments, the microorganism isnot methanotrophic.

The gaseous substrate comprises one or more of CO, CO₂, and H₂.Typically, the gaseous substrate comprises at least some amount of CO.In certain embodiments, the gaseous substrate does not comprise methane.However, in particular embodiments, the gaseous substrate may begenerated via methane reforming. The gaseous substrate may be or may bederived from an industrial waste gas, an industrial off gas, or syngas.

The animal feed is suitable for feeding to livestock or pets, including,but not limited to, beef cattle, dairy cattle, pigs, sheep, goats,horses, mules, donkeys, deer, buffalo/bison, llamas, alpacas, reindeer,camels, bantengs, gayals, yaks, chickens, turkeys, ducks, geese, quail,guinea fowl, squabs/pigeons, fish, shrimp, crustaceans, cats, dogs, androdents.

The animal feed may further comprise one or more excipients, such as acarbohydrate, fiber, fat, protein, vitamin, mineral, water, flavor,sweetener, antioxidant, enzyme, preservative, probiotic, or antibiotic.More generally, the excipient may be any substance added to themicrobial biomass to enhance or alter the form, properties, ornutritional content of the animal feed.

The invention further provides a method for producing the animal feedcomprising culturing a microorganism in the presence of a gaseoussubstrate to form microbial biomass and producing animal feed from themicrobial biomass. Generally, the gaseous substrate comprises one ormore of CO, CO₂, and H₂.

The method may comprise additional steps. For example, the method maycomprise a step of reducing the nucleic acid content of the microbialbiomass. The method may also comprise one or more steps of sterilizingthe microbial biomass, centrifuging the microbial biomass, and dryingthe microbial biomass. In particular, the drying may be spray drying orpaddle drying. Furthermore, the method may comprise blending themicrobial biomass with the excipient. The method may also compriseculturing the microorganism under fermentation conditions that maximizeproduction of microbial biomass.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered that microbial biomass produced from thefermentation of gaseous substrates, particularly gaseous substratescomprising one or more of CO, CO₂, and H₂, is particularly suitablesource of SCP for use in animal feed.

The invention provides animal feed comprising microbial biomass and atleast one excipient, wherein the microbial biomass comprises amicroorganism grown on a gaseous substrate, such as a gaseous substratecomprising one or more of CO, CO₂, and H₂.

A “microorganism” or “microbe” is a microscopic organism, especially abacterium, archea, virus, or fungus. The microorganism is typically abacterium. As used herein, recitation of “microorganism” should be takento encompass “bacterium.”

“Microbial biomass” refers biological material comprising microorganismcells. For example, microbial biomass may comprise or consist of a pureor substantially pure culture of a bacterium, archea, virus, or fungus.When initially separated from a fermentation broth, microbial biomassgenerally contains a large amount of water. This water may be removed orreduced by drying or processing the microbial biomass.

The microbial biomass may comprise any of the components listed in thefirst column of the table in Example 1. Notably, the microbial biomassof Example 1 comprises 15% moisture (water) by weight. Accordingly, thevalues listed in Example 1 refer to amounts of each component per amountof wet (i.e., non-dried) microbial biomass. Herein, the composition ofthe microbial biomass is described in terms of weight of a component perweight of wet (i.e., non-dried) microbial biomass. Of course, it is alsopossible to calculate the composition of the microbial biomass in termsof weight of a component per weight of dry microbial biomass.

The microbial biomass generally contains a large fraction of protein,such as more than 50% (50 g protein/100 g biomass), more than 60% (60 gprotein/100 g biomass), more than 70% (70 g protein/100 g biomass), ormore than 80% (80 g protein/100 g biomass) protein by weight. In apreferred embodiment, the microbial biomass comprises at least 72% (72 gprotein/100 g biomass) protein by weight. The protein fraction comprisesamino acids, including aspartic acid, alanine, arginine, cysteine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tyrosine, and/orvaline. In particular, the microbial biomass may comprise more than 10mg methionine/g biomass, more than 15 mg methionine/g biomass, more than20 mg methionine/g biomass, or more than 25 mg methionine/g biomass. Ina preferred embodiment, the microbial biomass comprises at least 17.6 mgmethionine/g biomass.

The microbial biomass may contain a number of vitamins, includingvitamins A (retinol), C, B1 (thiamine), B2 (riboflavin), B3 (niacin), B5(pantothenic acid), and/or B6 (pyridoxine).

The microbial biomass may contain relatively small amounts ofcarbohydrates and fats. For example, the microbial biomass may compriseless than 15% (15 g carbohydrate/100 g biomass), less than 10% (10 gcarbohydrate/100 g biomass), or less than 5% (5 g carbohydrate/100 gbiomass) of carbohydrate by weight. For example, the microbial biomassmay comprise less than 10% (10 g fat/100 g biomass), or less than 5% (5g fat/100 g biomass), less than 2% (2 g fat/100 g biomass), or less than1% (1 g fat/100 g biomass) of fat by weight.

The microorganism may classified based on functional characteristics.For example, the microorganism may be or may be derived from a C1-fixingmicroorganism, an anaerobe, an acetogen, an ethanologen, and/or acarboxydotroph. Table 1 provides a representative list of microorganismsand identifies their functional characteristics.

TABLE 1 C1-fixing Anaerobe Acetogen Ethanologen Autotroph CarboxydotrophMethanotroph Acetobacterium woodii + + + +/−¹ − +/−² − Alkalibaculumbacchii + + + + + + − Blautia producta + + + − + + − Butyribacteriummethylotrophicum + + + + + + − Clostridium aceticum + + + − + + −Clostridium autoethanogenum + + + + + + − Clostridiumcarboxidivorans + + + + + + − Clostridium coskatii + + + + + + −Clostridium drakei + + + − + + − Clostridium formicoaceticum + + + − + +− Clostridium ljungdahlii + + + + + + − Clostridium magnum + + + − ++/−³ − Clostridium ragsdalei + + + + + + − Clostridiumscatologenes + + + − + + − Eubacterium limosum + + + − + + − Moorellathermautotrophica + + + + + + − Moorella thermoacetica (formerly + + +−⁴ + + − Clostridium thermoaceticum) Oxobacter pfennigii + + + − + + −Sporomusa ovata + + + − + +/−⁵ − Sporomusa silvacetica + + + − + +/−⁶ −Sporomusa sphaeroides + + + − + +/−⁷ − Thermoanaerobacter kiuvi + + +− + − − ¹ Acetobacterium woodi can produce ethanol from fructose, butnot from gas. ²It has been reported that Acetobacterium woodi can growon CO, but the methodology is questionable. ³It has not beeninvestigated whether Clostridium magnum can grow on CO. ⁴One strain ofMoorella thermoacetica, Moorella sp. HUC22-1, has been reported toproduce ethanol from gas. ⁵It has not been investigated whetherSporomusa ovata can grow on CO. ⁶It has not been investigated whetherSporomusa silvacetica can grow on CO. ⁷It has not been investigatedwhether Sporomusa sphaeroides can grow on CO.

“C1” refers to a one-carbon molecule, for example, CO or CO₂.“C1-oxygenate” refers to a one-carbon molecule that also comprises atleast one oxygen atom, for example, CO or CO₂. “C1-carbon source” refersa one carbon-molecule that serves as a partial or sole carbon source forthe microorganism. For example, a C1-carbon source may comprise one ormore of CO, CO₂, or CH₂O₂. Preferably, the C1-carbon source comprisesone or both of CO and CO₂. A “C1-fixing microorganism” is amicroorganism that has the ability to produce one or more products froma C1-carbon source. Typically, the microorganism is a C1-fixingbacterium. In a preferred embodiment, the microorganism is or is derivedfrom a C1-fixing microorganism identified in Table 1.

An “anaerobe” is a microorganism that does not require oxygen forgrowth. An anaerobe may react negatively or even die if oxygen ispresent above a certain threshold. Typically, the microorganism is ananaerobe (i.e., is anaerobic). In a preferred embodiment, themicroorganism is or is derived from an anaerobe identified in Table 1.

An “acetogen” is a microorganism that produces or is capable ofproducing acetate (or acetic acid) as a product of anaerobicrespiration. Typically, acetogens are obligately anaerobic bacteria thatuse the Wood-Ljungdahl pathway as their main mechanism for energyconservation and for synthesis of acetyl-CoA and acetyl-CoA-derivedproducts, such as acetate (Ragsdale, Biochim Biophys Acta, 1784:1873-1898, 2008). Acetogens use the acetyl-CoA pathway as a (1)mechanism for the reductive synthesis of acetyl-CoA from CO₂, (2)terminal electron-accepting, energy conserving process, (3) mechanismfor the fixation (assimilation) of CO₂ in the synthesis of cell carbon(Drake, Acetogenic Prokaryotes, In: The Prokaryotes, 3^(rd) edition, p.354, New York, N.Y., 2006). All naturally occurring acetogens areC1-fixing, anaerobic, autotrophic, and non-methanotrophic. In apreferred embodiment, the microorganism is an acetogen. In a preferredembodiment, the microorganism is or is derived from an acetogenidentified in Table 1.

An “ethanologen” is a microorganism that produces or is capable ofproducing ethanol. In a preferred embodiment, the microorganism is anethanologen. In a preferred embodiment, the microorganism is or isderived from an ethanologen identified in Table 1.

An “autotroph” is a microorganism capable of growing in the absence oforganic carbon. Instead, autotrophs use inorganic carbon sources, suchas CO and/or CO₂. In a preferred embodiment, the microorganism is anautotroph. In a preferred embodiment, the microorganism is or is derivedfrom an autotroph identified in Table 1.

A “carboxydotroph” is a microorganism capable of utilizing CO as a solesource of carbon. In a preferred embodiment, the microorganism is acarboxydotroph. In a preferred embodiment, the microorganism is or isderived from a carboxydotroph identified in Table 1.

In certain embodiments, the microorganism does not consume certainsubstrates, such as methane or methanol. In one embodiment, themicroorganism is not a methanotroph and/or is not a methylotroph.

Preferably, the microorganism is Gram-positive. Most prior work on theuse of SCP in animal feed involved Gram-negative species, such asMethylophilus methylotrophicus, Methylophilus clara, and Methylophilusmethanica. However, Gram-negative bacteria often produce or containendotoxins that make their use in animal feed problematic. Becauseendotoxins are the part of cellular components of some of theGram-negative bacteria and are not released into the medium by theliving bacterial cell, their removal is somewhat difficult. Theirformation can only be prevented by genetic engineering, where theactivity of genes controlling the formation of the unwanted toxins canbe modified or suppressed. This may be a difficult task to achieve, asthey are integral structural components of the bacterial cell wall(Ravindra, Biotechnol Adv, 18: 459-479, 2000).

More broadly, the microorganism may be or may be derived from any genusor species identified in Table 1. For example, the microorganism may bea member of the genus Clostridium.

In a preferred embodiment, the microorganism is or is derived from thecluster of Clostridia comprising the species Clostridiumautoethanogenum, Clostridium ljungdahlii, and Clostridium ragsdalei.These species were first reported and characterized by Abrini, ArchMicrobiol, 161: 345-351, 1994 (Clostridium autoethanogenum), Tanner, IntJ System Bacteriol, 43: 232-236, 1993 (Clostridium ljungdahlii), andHuhnke, WO 2008/028055 (Clostridium ragsdalei).

These three species have many similarities. In particular, these speciesare all C1-fixing, anaerobic, acetogenic, ethanologenic, andcarboxydotrophic members of the genus Clostridium. These species havesimilar genotypes and phenotypes and modes of energy conservation andfermentative metabolism. Moreover, these species are clustered inclostridial rRNA homology group I with 16S rRNA DNA that is more than99% identical, have a DNA G+C content of about 22-30 mol %, aregram-positive, have similar morphology and size (logarithmic growingcells between 0.5-0.7×3-5 μm), are mesophilic (grow optimally at 30-37°C.), have similar pH ranges of about 4-7.5 (with an optimal pH of about5.5-6), lack cytochromes, and conserve energy via an Rnf complex. Also,reduction of carboxylic acids into their corresponding alcohols has beenshown in these species (Perez, Biotechnol Bioeng, 110:1066-1077, 2012).Importantly, these species also all show strong autotrophic growth onCO-containing gases, produce ethanol and acetate (or acetic acid) asmain fermentation products, and produce small amounts of 2,3-butanedioland lactic acid under certain conditions.

However, these three species also have a number of differences. Thesespecies were isolated from different sources: Clostridiumautoethanogenum from rabbit gut, Clostridium ljungdahlii from chickenyard waste, and Clostridium ragsdalei from freshwater sediment. Thesespecies differ in utilization of various sugars (e.g., rhamnose,arabinose), acids (e.g., gluconate, citrate), amino acids (e.g.,arginine, histidine), and other substrates (e.g., betaine, butanol).Moreover, these species differ in auxotrophy to certain vitamins (e.g.,thiamine, biotin). These species have differences in nucleic and aminoacid sequences of Wood-Ljungdahl pathway genes and proteins, althoughthe general organization and number of these genes and proteins has beenfound to be the same in all species (Kopke, Curr Opin Biotechnol, 22:320-325, 2011).

Thus, in summary, many of the characteristics of Clostridiumautoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei arenot specific to that species, but are rather general characteristics forthis cluster of C1-fixing, anaerobic, acetogenic, ethanologenic, andcarboxydotrophic members of the genus Clostridium. However, since thesespecies are, in fact, distinct, the genetic modification or manipulationof one of these species may not have an identical effect in another ofthese species. For instance, differences in growth, performance, orproduct production may be observed.

The microorganism may also be or be derived from an isolate or mutant ofClostridium autoethanogenum, Clostridium ljungdahlii, or Clostridiumragsdalei. Isolates and mutants of Clostridium autoethanogenum includeJA1-1 (DSM10061) (Abrini, Arch Microbiol, 161: 345-351, 1994), LBS1560(DSM19630) (WO 2009/064200), and LZ1561 (DSM23693). Isolates and mutantsof Clostridium ljungdahlii include ATCC 49587 (Tanner, Int J SystBacteriol, 43: 232-236, 1993), PETCT (DSM13528, ATCC 55383), ERI-2 (ATCC55380) (U.S. Pat. No. 5,593,886), C-01 (ATCC 55988) (U.S. Pat. No.6,368,819), 0-52 (ATCC 55989) (U.S. Pat. No. 6,368,819), and OTA-1(Tirado-Acevedo, Production of bioethanol from synthesis gas usingClostridium ljungdahlii, PhD thesis, North Carolina State University,2010). Isolates and mutants of Clostridium ragsdalei include PI 1 (ATCCBAA-622, ATCC PTA-7826) (WO 2008/028055).

The term “derived from” refers to a microorganism is modified or adaptedfrom a different (e.g., a parental or wild-type) microorganism, so as toproduce a new microorganism. Such modifications or adaptations typicallyinclude insertion, deletion, mutation, or substitution of nucleic acidsor genes.

“Substrate” refers to a carbon and/or energy source for themicroorganism. Typically, the substrate is gaseous and comprises aC1-carbon source, for example, CO or CO₂. Preferably, the substratecomprises a C1-carbon source of CO or CO+CO₂. The substrate may furthercomprise other non-carbon components, such as H₂, N₂, or electrons.

The substrate generally comprises at least some amount of CO, such asabout 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mol % CO. Thesubstrate may comprise a range of CO, such as about 20-80, 30-70, or40-60 mol % CO. Preferably, the substrate comprises about 40-70 mol % CO(e.g., steel mill or blast furnace gas), about 20-30 mol % CO (e.g.,basic oxygen furnace gas), or about 15-45 mol % CO (e.g., syngas). Insome embodiments, the substrate may comprise a relatively low amount ofCO, such as about 1-10 or 1-20 mol % CO. The microorganism typicallyconverts at least a portion of the CO and/or in the substrate to aproduct. In some embodiments, the substrate comprises no orsubstantially no CO.

The substrate may comprise some amount of H₂. For example, the substratemay comprise about 1, 2, 5, 10, 15, 20, or 30 mol % H₂. In someembodiments, the substrate may comprise a relatively high amount of H₂,such as about 60, 70, 80, or 90 mol % H₂. In further embodiments, thesubstrate comprises no or substantially no H₂.

The substrate may comprise some amount of CO₂. For example, thesubstrate may comprise about 1-80 or 1-30 mol % CO₂. In someembodiments, the substrate may comprise less than about 20, 15, 10, or 5mol % CO₂. In another embodiment, the substrate comprises no orsubstantially no CO₂.

In some embodiments, the substrate does not comprise methane ormethanol.

Although the substrate is typically gaseous, the substrate may also beprovided in alternative forms. For example, the substrate may bedissolved in a liquid saturated with a CO-containing gas using amicrobubble dispersion generator. By way of further example, thesubstrate may be adsorbed onto a solid support.

The substrate and/or C1-carbon source may be or may be derived from awaste or off gas obtained as a byproduct of an industrial process orfrom some other source, such as from automobile exhaust fumes or biomassgasification. In certain embodiments, the industrial process is selectedfrom the group consisting of ferrous metal products manufacturing, suchas a steel mill manufacturing, non-ferrous products manufacturing,petroleum refining processes, coal gasification, electric powerproduction, carbon black production, ammonia production, methanolproduction, and coke manufacturing. In these embodiments, the substrateand/or C1-carbon source may be captured from the industrial processbefore it is emitted into the atmosphere, using any convenient method.

The substrate and/or C1-carbon source may be or may be derived fromsyngas, such as syngas obtained by gasification of coal or refineryresidues, gasification of biomass or lignocellulosic material, orreforming of natural gas. In another embodiment, the syngas may beobtained from the gasification of municipal solid waste or industrialsolid waste.

In connection with substrates and/or C1-carbon sources, the term“derived from” refers to a substrate and/or C1-carbon source that issomehow modified or blended. For example, the substrate and/or C1-carbonsource may be treated to add or remove certain components or may beblended with streams of other substrates and/or C1-carbon sources.

The composition of the substrate may have a significant impact on theefficiency and/or cost of the reaction. For example, the presence ofoxygen (02) may reduce the efficiency of an anaerobic fermentationprocess. Depending on the composition of the substrate, it may bedesirable to treat, scrub, or filter the substrate to remove anyundesired impurities, such as toxins, undesired components, or dustparticles, and/or increase the concentration of desirable components.

The invention further provides a method for producing animal feed,comprising culturing a microorganism in the presence of a gaseoussubstrate comprising one or more of CO, CO₂, and H₂ to form microbialbiomass and producing animal feed from the microbial biomass.

Typically, the culture is performed in a bioreactor. The term“bioreactor” includes a culture/fermentation device consisting of one ormore vessels, towers, or piping arrangements, such as a continuousstirred tank reactor (CSTR), immobilized cell reactor (ICR), trickle bedreactor (TBR), bubble column, gas lift fermenter, static mixer, or othervessel or other device suitable for gas-liquid contact. In someembodiments, the bioreactor may comprise a first growth reactor and asecond culture/fermentation reactor. The substrate may be provided toone or both of these reactors. As used herein, the terms “culture” and“fermentation” are used interchangeably. These terms encompass both thegrowth phase and product biosynthesis phase of the culture/fermentationprocess.

The culture is generally maintained in an aqueous culture medium thatcontains nutrients, vitamins, and/or minerals sufficient to permitgrowth of the microorganism. Preferably the aqueous culture medium is ananaerobic microbial growth medium, such as a minimal anaerobic microbialgrowth medium. Suitable media are well known in the art.

The culture/fermentation should desirably be carried out underappropriate conditions for production of the target product. Reactionconditions to consider include pressure (or partial pressure),temperature, gas flow rate, liquid flow rate, media pH, media redoxpotential, agitation rate (if using a continuous stirred tank reactor),inoculum level, maximum gas substrate concentrations to ensure that gasin the liquid phase does not become limiting, and maximum productconcentrations to avoid product inhibition. In particular, the rate ofintroduction of the substrate may be controlled to ensure that theconcentration of gas in the liquid phase does not become limiting, sinceproducts may be consumed by the culture under gas-limited conditions.

Herein, microbial biomass itself is considered a target product.However, the microorganism also produce one or more other products ofvalue. For instance, Clostridium autoethanogenum produces or can beengineered to produce ethanol (WO 2007/117157), acetate (WO2007/117157), butanol (WO 2008/115080 and WO 2012/053905), butyrate (WO2008/115080), 2,3-butanediol (WO 2009/151342), lactate (WO 2011/112103),butene (WO 2012/024522), butadiene (WO 2012/024522), methyl ethyl ketone(2-butanone) (WO 2012/024522 and WO 2013/185123), ethylene (WO2012/026833), acetone (WO 2012/115527), isopropanol (WO 2012/115527),lipids (WO 2013/036147), 3-hydroxypropionate (3-HP) (WO 2013/180581),isoprene (WO 2013/180584), fatty acids (WO 2013/191567), 2-butanol (WO2013/185123), 1,2-propanediol (WO 2014/0369152), and 1-propanol (WO2014/0369152).

Operating a bioreactor at elevated pressures allows for an increasedrate of gas mass transfer from the gas phase to the liquid phase.Accordingly, it is generally preferable to perform theculture/fermentation at pressures higher than atmospheric pressure.Also, since a given gas conversion rate is, in part, a function of thesubstrate retention time and retention time dictates the required volumeof a bioreactor, the use of pressurized systems can greatly reduce thevolume of the bioreactor required and, consequently, the capital cost ofthe culture/fermentation equipment. This, in turn, means that theretention time, defined as the liquid volume in the bioreactor dividedby the input gas flow rate, can be reduced when bioreactors aremaintained at elevated pressure rather than atmospheric pressure. Theoptimum reaction conditions will depend partly on the particularmicroorganism used. Also, since a given gas conversion rate is in part afunction of substrate retention time and achieving a desired retentiontime in turn dictates the required volume of a bioreactor, the use ofpressurized systems can greatly reduce the volume of the bioreactorrequired, and consequently the capital cost of the fermentationequipment.

The culturing of the microorganism may be performed under fermentationconditions that maximize production of microbial biomass. The method mayalso comprise culturing the microorganism under fermentation conditionsthat maximize production of or selectivity to microbial biomass.Maximizing selectivity to biomass requires operation at maximal specificgrowth rates or maximal microorganism dilution rate. However, operationat high microorganism dilution rates also reduces the cell concentrationin the culture which hampers separations. Also, cell concentration is akey requirement for high reactor productivity. Specific growth rates ormicroorganism dilution rates of >1/day should be targeted, with rates of2/day being closer to the optimum.

In a two reactor system, biomass production rates are maximized byhaving high biomass production rates in both the first and secondreactor. This can be achieved by either having (1) low cell viability or(2) fast specific growth rates in the second reactor. Low cell viabilitymay be achieved from the toxicity of high product titers and may not bedesirable. Fast specific growth rates may be achieved by operating witheven higher values of microorganism dilution rate in the second reactorcompared to the first reactor.

This relationship is captured by the following equation:μ₂=D_(w2)−D_(w1)*(X₁/X₂)*(V₁/V₂), where μ₂ is the specific growth ratein the second reactor in a two reactor system which will need to bemaximized to increase selectivity to biomass, D_(w2) and D_(w1) are themicroorganism dilution rates in the second and first reactors in a tworeactor system, respectively, X₂ and X₁ are the biomass titers in thesecond and first reactors in a two reactor system, respectively, and V₂and V₁ are the reactor volumes in the second and first reactors in a tworeactor system, respectively.

According to this equation, to maximize the selectivity to biomass in asecond reactor, the microorganism dilution rate in the second reactor,D_(w2), will need to be increased to achieve a specific growth rate, μ₂,in the second reactor of >0.5/day, ideally targeting 1-2/day.

Products may be separated or purified from a fermentation broth usingany method or combination of methods known in the art, including, forexample, fractional distillation, evaporation, pervaporation, gasstripping, phase separation, and extractive fermentation, including forexample, liquid-liquid extraction. In certain embodiments, products arerecovered from the fermentation broth by continuously removing a portionof the broth from the bioreactor, separating microbial cells from thebroth (conveniently by filtration), and recovering one or more targetproducts from the broth. Alcohols and/or acetone may be recovered, forexample, by distillation. Acids may be recovered, for example, byadsorption on activated charcoal. Cell-free permeate remaining afterproducts have been removed is also preferably returned to thebioreactor. Additional nutrients (such as B vitamins) may be added tothe cell-free permeate to replenish the medium before it is returned tothe bioreactor.

The method of the invention may further comprise additional separation,processing, or treatment steps. For example, the method may comprisesterilizing the microbial biomass, centrifuging the microbial biomass,and/or drying the microbial biomass. In certain embodiments, themicrobial biomass is dried using spray drying or paddle drying. Themethod may also comprise reducing the nucleic acid content of themicrobial biomass using any method known in the art, since intake of adiet high in nucleic acid content may result in the accumulation ofnucleic acid degradation products and/or gastrointestinal distress.Furthermore, the method may comprise blending or combining the microbialbiomass with one or more excipients.

The animal feed is suitable for feeding to animals, such as livestock orpets. In particular, the animal feed may be suitable for feeding to oneor more of beef cattle, dairy cattle, pigs, sheep, goats, horses, mules,donkeys, deer, buffalo/bison, llamas, alpacas, reindeer, camels,bantengs, gayals, yaks, chickens, turkeys, ducks, geese, quail, guineafowl, squabs/pigeons, fish, shrimp, crustaceans, cats, dogs, androdents. The composition of the animal feed may be tailored to thenutritional requirements of different animals.

Generally, the animal feed comprises at least one excipient. Herein,“excipient” refers to any substance that may be added to the microbialbiomass to enhance or alter the form, properties, or nutritional contentof the animal feed. For example, the excipient may comprise one or moreof a carbohydrate, fiber, fat, protein, vitamin, mineral, water, flavor,sweetener, antioxidant, enzyme, preservative, probiotic, or antibiotic.In some embodiments, the excipient may be hay, straw, silage, grains,oils or fats, or other plant material.

The excipient may be any feed ingredient identified in Chiba, Section18: Diet Formulation and Common Feed Ingredients, Animal NutritionHandbook, 3^(rd) revision, pages 575-633, 2014, including alfalfa,animal fat, bakery waste, barley, beet pulp, bermudagrass, blood meal orplasma, brewer's grain, brewer's yeast, bromegrass, buckwheat,canarygrass, canola, casein, citrus pulp, clover, coconut, corn, corncob, cottonseed, feather meal, fescue, fish meal or solubles, hominy,meat or bone meal, milk, millet, molasses, oats, orchardgrass, peas,peanuts, poultry meal, rice, rye, ryegrass, safflower, sesame, sorghum,soybean, sunflower, timothy, triticale, urea, wheat, whey, or yeast.

EXAMPLES

The following examples further illustrate the invention but, of course,should not be construed to limit its scope in any way.

Example 1

This example describes the composition of C. autoethanogenum DSM23693microbial biomass.

Component Result Unit Testing Method Calories (calculation) 329 kcal/100g 21 CFR Part 101 Calories from fat ND kcal/100 g 21 CFR Part 101(calculation) Total carbohydrates 10 g/100 g 21 CFR Part 101(calculation) Vitamin A (retinol) ND IU/100 g AOAC 2001.13 Calcium 42mg/100 g AOAC 2011.14 Iron 29 mg/100 g AOAC 2011.14 Sodium 170 mg/100 gAOAC 2011.14 Copper 0.525 mg/Kg SW6010C/SW3061 Magnesium 193 mg/KgSW6010C/SW3065 Manganese <4.7 mg/Kg SW6010C/SW3066 Phosphorus 6720 mg/KgSW6010C/SW3066 Potassium 6520 mg/Kg SW6010C/SW3066 Selenium 14.3 mg/KgSW6010C/SW3066 Sodium 1960 mg/Kg SW6010C/SW3066 Zinc 53 mg/KgSW6010C/SW3066 Ash 2.6 g/100 g AOAC 923.03 Moisture 15 g/100 g AOAC927.05/950.46 Total sugar ND g/100 g AOAC 982.14 Total dietary fiber 9.1g/100 g AOAC 2011.25 mod Protein 72 g/100 g AOAC 992.15/992.23Cholesterol ND mg/100 g AOAC 994.1 Monounsaturated fat ND g/100 g AOAC996.06 Polyunsaturated fat ND g/100 g AOAC 996.06 Saturated fat ND g/100g AOAC 996.06 Total fat ND g/100 g AOAC 996.06 Trans fat ND g/100 g AOAC996.06 Vitamin C ND mg/100 g JAFC (2003) B1 (thiamine) 0.07 mg/100 gVitamin¹ B2 (riboflavin) 3.53 mg/100 g Vitamin¹ B3 (niacin) 7.44 mg/100g Vitamin¹ B5 (pantothenic acid) 0.12 mg/100 g Vitamin¹ B6 (pyridoxine)0.532 mg/100 g Vitamin¹ Total amino acids Aspartic acid 78.5 mg/g Totalamino acid² Alanine 44 mg/g Total amino acid² Arginine 27.9 mg/g Totalamino acid² Cystine 6.35 mg/g Total amino acid² Glutamic acid 73.4 mg/gTotal amino acid² Glycine 31.2 mg/g Total amino acid² Histidine 10.2mg/g Total amino acid² Isoleucine 43.9 mg/g Total amino acid² Leucine48.7 mg/g Total amino acid² Lysine 66.2 mg/g Total amino acid²Methionine 17.6 mg/g Total amino acid² Phenylalanine 25.8 mg/g Totalamino acid² Proline 21.7 mg/g Total amino acid² Serine 27.8 mg/g Totalamino acid² Threonine 34.7 mg/g Total amino acid² Tyrosine 29.4 mg/gTotal amino acid² Valine 41.9 mg/g Total amino acid² ¹AOAC 944.13, AOAC960.46, AOAC 945.74, AOAC 961.15, AOAC 940.33, AOAC 942.23, AOAC 953.17,AOAC 957.17 ²Methods used: AOAC 944.13, AOAC 960.46, AOAC 945.74, AOAC961.15, AOAC 940.33, AOAC 942.23, AOAC 953.17, AOAC 957.17, AOAC 988.15,R. Schuster, “Determination of Amino Acids in Biological,Pharmaceutical, Plant and Food Samples by Automated PrecolumnDerivatization and HPLC”, Journal of Chromatography, 1988, 431, 271-284.Henderson, J. W., Brooks, A., “Improved Amino Acid Methods using AgilentZorbax Eclipse Plus C18 Columns for a Variety of Agilent LCInstrumentation and Separation Goals,” Agilent Application Note5990-4547 (2010)., Henderson, J. W., Ricker, R. D. Bidlingmeyer, B. A.,Woodward, C., “Rapid, Accurate, Sensitive, and Reproducible HPLCAnalysis of Amino Acids, Amino Acid Analysis Using Zorbax Eclipse-AAAcolumns and the Agilent 1100 HPLC,” Agilent Publication, 2000. nt = nottested ND = not detected (below the detection limit of the method) <=element not detected; value shown is the limit of detection of themethod

Example 2

This example compares the amino acid composition of C. autoethanogenumDSM23693 biomass to the amino acid compositions of other types of feedprotein supplements.

Herring White Type C. autoethanogenum Soybean Fish Fish DSM23693 biomassMeal* Meal* Meal* Unit Alanine 3.2 na na na g per 100 g protein Arginine2.0 3.2 4.1 4.2 g per 100 g protein Aspartic acid 5.7 na na na g per 100g protein Cystine 0.5 1.3 2.3 2.9 g per 100 g protein Glutamic acid 5.3na na na g per 100 g protein Glycine 2.2 1.9 6.5 4.3 g per 100 g proteinHistidine 0.7 1.1 1.3 1.7 g per 100 g protein Isoleucine 3.2 2.2 2.4 3.2g per 100 g protein Leucine 3.5 3.4 4.2 5.4 g per 100 g protein Lysine4.8 2.9 4.5 5.5 g per 100 g protein Methionine 1.3 0.6 1.7 2.2 g per 100g protein Phenylalanine 1.9 2.2 2.1 2.8 g per 100 g protein Proline 1.6na na na g per 100 g protein Serine 2.0 2.5 3.1 2.8 g per 100 g proteinThreonine 2.5 1.9 2.5 3.1 g per 100 g protein Tryptophan na 0.6 0.6 0.8g per 100 g protein Tyrosine 2.1 1.6 1.7 2.3 g per 100 g protein Valinena 2.3 2.9 3.9 g per 100 g protein Total Crude 72.0 45.0 65.0 72.0 g per100 g Protein Macronutrients comparison Moisture 15 11 10 8 g per 100 gTotal protein 72 46 65 72 g per 100 g Crude fat ND 1 5 9 g per 100 g Ash3 6 20 10 g per 100 g Crude fiber 9 6 0 0 g per 100 g na = not available*FAO Fisheries Technical Paper - 142, (1986). Retrieved 2015. ND = notdetected (below the detection limit of the method)

Example 3

This example describes general methods for culturing C. autoethanogenumand C. ljungdahlii. Such methods are also well known in the art.

C. autoethanogenum DSM10061 and DSM23693 (a derivate of DSM10061) and C.ljungdahlii DSM13528 were sourced from DSMZ (The German Collection ofMicroorganisms and Cell Cultures, Inhoffenstraβe 7 B, 38124Braunschweig, Germany).

Strains were grown at 37° C. in PETC medium at pH 5.6 using standardanaerobic techniques (Hungate, Methods Microbiol, 3B: 117-132, 1969;Wolfe, Adv Microbiol Physiol, 6: 107-146, 1971). Fructose (heterotrophicgrowth) or 30 psi CO-containing steel mill gas (collected from NewZealand Steel site in Glenbrook, NZ; composition: 44% CO, 32% N₂, 22%CO₂, 2% H₂) in the headspace (autotrophic growth) was used as substrate.For solid media, 1.2% bacto agar (BD, Franklin Lakes, N.J. 07417, USA)was added.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein. The reference to any prior art in this specification is not, andshould not be taken as, an acknowledgement that that prior art formspart of the common general knowledge in the field of endeavour in anycountry.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein. Variationsof those preferred embodiments may become apparent to those of ordinaryskill in the art upon reading the foregoing description. The inventorsexpect skilled artisans to employ such variations as appropriate, andthe inventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

1. Animal feed comprising microbial biomass and at least one excipient,wherein the microbial biomass comprises a microorganism grown on agaseous substrate comprising one or more of CO, CO₂, and H₂.
 2. Theanimal feed of claim 1, wherein the microorganism is Gram-positive. 3.The animal feed of claim 1, wherein the microorganism is acetogenicand/or carboxydotrophic.
 4. The animal feed of claim 1, wherein themicroorganism is anaerobic.
 5. The animal feed of claim 1, wherein themicroorganism is a member of the genus Clostridium.
 6. The animal feedof claim 1, wherein the microorganism is or is derived from Clostridiumautoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, orClostridium coskatii.
 7. The animal feed of claim 1, wherein themicroorganism is not methanotrophic.
 8. The animal feed of claim 1,wherein the gaseous substrate comprises CO.
 9. The animal feed of claim1, wherein the gaseous substrate does not comprise methane.
 10. Theanimal feed of claim 1, wherein the gaseous substrate is or is derivedfrom industrial waste gas, industrial off gas, or syngas.
 11. The animalfeed of claim 1, wherein the animal feed is suitable for feeding tolivestock or pets.
 12. The animal feed of claim 1, wherein the animalfeed is suitable for feeding to one or more of beef cattle, dairycattle, pigs, sheep, goats, horses, mules, donkeys, deer, buffalo/bison,llamas, alpacas, reindeer, camels, bantengs, gayals, yaks, chickens,turkeys, ducks, geese, quail, guinea fowl, squabs/pigeons, fish, shrimp,crustaceans, cats, dogs, and rodents.
 13. An animal feed of claim 1,wherein the excipient comprises one or more of a carbohydrate, fiber,fat, protein, vitamin, mineral, water, flavor, sweetener, antioxidant,enzyme, preservative, probiotic, or antibiotic.
 14. An animal feed ofclaim 1, wherein the excipient comprises one or more of alfalfa, animalfat, bakery waste, barley, beet pulp, bermudagrass, blood meal orplasma, brewer's grain, brewer's yeast, bromegrass, buckwheat,canarygrass, canola, casein, citrus pulp, clover, coconut, corn, corncob, cottonseed, feather meal, fescue, fish meal or solubles, hominy,meat or bone meal, milk, millet, molasses, oats, orchardgrass, peas,peanuts, poultry meal, rice, rye, ryegrass, safflower, sesame, sorghum,soybean, sunflower, timothy, triticale, urea, wheat, whey, or yeast. 15.A method for producing the animal feed of claim 1, comprising culturinga microorganism in the presence of a gaseous substrate comprising one ormore of CO, CO₂, and H₂ to form microbial biomass and producing animalfeed from the microbial biomass.
 16. The method of claim 15, wherein themethod further comprises reducing the nucleic acid content of themicrobial biomass.
 17. The method of claim 15, wherein the methodfurther comprises one or more steps selected from the group consistingof sterilizing the microbial biomass, centrifuging the microbialbiomass, and drying the microbial biomass.
 18. The method of claim 17,wherein the drying is spray drying or paddle drying.
 19. The method ofclaim 17, wherein the method further comprises blending the microbialbiomass with the excipient.
 20. The method of claim 15, whereinculturing is performed under fermentation conditions that maximizeproduction of microbial biomass.